Anti-herpesviral agent

ABSTRACT

An antiviral agent capable of disrupting the association of two viral proteins required for DNA replication in herpesviruses. The agents disrupt the association of UL8 and POL in HSV-1 or the association of equivalent homologues of these proteins in other herpesviruses (for example UL 102 and UL54 in HCMV). Suitable agents are peptides which mimic the C-terminal or C-proximal portion of UL8 (or its homologues) for example the peptide IELVFTGVLAGVWGEGGKFV. Peptidomimetic compounds of such peptides are also suitable anti-viral agents. An assay to test for agents capable of disrupting association of POL and UL8 (or homologues thereof) is also described.

This application is the U.S. national phase application of PCTInternational Application No. PCT/GB97/02025 filed Jul. 28, 1997.

The present invention relates to an anti-viral agent effective againstherpesviruses and to an assay for screening for other suitableanti-viral agents.

Herpesviruses include Herpes Simplex Virus types 1 and 2 (HSV-1 andHSV-2), Human Cytomegalovirus (HCMV), Epstein-Barr Virus (EBV) andEquine herpesviruses 1 and 4 (EHV-1 and EHV-4). The term “Herpesvirus”is used herein to refer to any virus of the herpesvirus family,including viruses in the α group (e.g. HSV 1 & 2, EHV 1 & 4), the βgroup (e.g. HCMV) and in the γ group (e.g. EBV).

Infections due to HSV have been successfully treated for many yearsthrough use of the drug acyclovir, a nucleoside analogue. Acyclovir isrelatively non-toxic to the human host since it does not adverselyaffect the activity of the mammalian homologue of the targeted viralprotein. However, similar low toxicity regimes for treating allherpesviruses have not yet been found. Whilst HCMV is treatable via useof the drug gancyclovir (Coen, 1992) the application of this drug islimited by its toxicity, poor bioavailability and the emergence ofdrug-resistant variants (reviewed by Coen 1992; Haffey & Field 1995;Filley et al 1995). A low-toxicity treatment for HCMV is particularly ofinterest as infection by this virus can cause congenital abnormalitiesin the newborn exposed to the virus by maternal transmission, and isalso extremely problematic to immunocompromised patients, for examplepatients suffering from AIDS, or those on immunosuppressive therapy forcancer or following organ transplant.

The genome of herpes simplex virus type 1 (HSV-1) encodes seven proteinsessential for origin dependent viral DNA synthesis (Wu et al., 1988).The genes encoding these proteins, and their protein products, are knownin the art as UL5, UL8, UL9, UL29, UL30, UL42 and UL52. (McGeoch et al.,1988). Frequently the names of the genes are italicized, eg UL5, toavoid possible ambiguities. The UL30 protein, the catalytic subunit ofthe heterodimeric HSV-1 DNA polymerase, is also known as POL. Homologuesof all seven genes have been identified in other alphaherpesviruses andhuman herpesviruses 6 and 7 (HHV-6 and HHV-7). Other beta- andgammaherpesviruses encode homologues of all these proteins except UL9.For convenience the terminology of the HSV-1 proteins will be used torefer not only to that particular protein but also its equivalent inother herpesviruses. Thus, as used herein the term “UL8” refers not onlyto UL8 of HSV-1 itself, but also to the HCMV homolgue UL102 and toequivalent homologues in other herpesviruses. Similarly, as used hereinthe term “POL” (or “UL30”) refers not only to POL of HSV-1 itself, butalso to the HCMV homolgue UL54 and to equivalent homolgues in otherherpesviruses.

The functions of these proteins and their interactions may be summarisedas follows. The UL9 product is an origin-binding protein (OBP) and theUL29 product (ICP8) a single-stranded DNA binding protein. These twoproteins can interact via the C-terminus of UL9 (Boehmer and Lehman,1993; Boehmer et al., 1994). The UL30 protein (POL) and UL42 proteinscomprise the catalytic and accessory components, respectively, of adimeric DNA polymerase (reviewed by Challberg, 1991; Weller, 1991) andinteract via residues at or near the C-terminus of POL (Digard & Coen,1990; Digard et al., 1993, 1995; Marsden at al., 1994; Stow et al.,1993; Tenney et al., 1993). The UL5, UL8 and UL52 proteins form atrimeric complex that exhibits both DNA helicase and DNA primaseactivities (Dodson et al., 1989; Crute et al., 1989). The UL5 protein islargely responsible for DNA helicase activity (Gorbalenya et al., 1989;Zhu & Weller, 1992), and the UL52 protein contributes an essential rolein DNA priming (Klinedinst & Challberg, 1994; Dracheva et al., 1995) andthese two proteins can form a stable subassembly that retains bothfunctions (Calder & Stow, 1990; Dodson & Lehman, 1991; Crute et al.,1991). The UL8 component has auxiliary effects on the DNA primaseactivity, stimulating primer synthesis and/or utilization on anatural-sequence single-stranded DNA template (Sherman et al., 1992;Tenney et al., 1994), and is also required for efficient nuclear entryof the trimeric complex. (Calder et al., 1992; Marsden et al., 1996).UL8 is capable of binding separately to the UL5 and UL52 proteins andcan also interact specifically with UL9 (McLean et al., 1994). Thelatter interaction with OBP may serve to recruit the helicase-primaseinto an initiation complex at the viral origins.

Further evidence for the occurrence of multiple interactions between DNAreplication proteins has been provided by immunofluorescenceexperiments. In cells infected with HSV-1 in the presence of inhibitorsof viral DNA synthesis UL29 (ICP8) localises to punctate structureswithin the nucleus termed “pre-replicative sites” (Quinlan et al.,1984). The requirement for each of the DNA replication proteins in theformation of these sites has been studied by the use of viral mutantswith defects in individual replication proteins (Liptak et al., 1996;Lukonis et al., 1996). It was observed that proteins UL5, UL8, UL9 andUL52 are all necessary for the localisation of UL29 (ICP8) intopre-replicative sites and that mutants with defects in any of the othersix DNA replication genes are affected in the ability of POL to localizeto these sites. Although these data suggest that the DNA polymeraseholoenzyme is the last component to be recruited (Liptak et al., 1996)they do not identify the specific interactions involved in this event.

It has now been found that the protein UL8 interacts with POL. Further,it has been found that disruption of the POL/UL8 interaction ispossible. Examples of molecules, monoclonal antibodies and peptides thatspecifically disrupt the interaction have been identified.

The present invention provides an anti-viral agent capable of combattingreplication of a herpesvirus by interfering with the association of UL8and POL (as defined above).

Both the UL8 and POL proteins of HSV-1 have been previously described inthe literature (e.g. Parry et al., 1993; Gottleib et al., 1990).

Furthermore the amino acid/DNA sequences of UL8 and POL from HSV-1 areavailable from publically accessible Genbank and EMBL databases underNos. P10192/M19120 and P04293/M12356 (and several other entries),respectively.

The UL8/POL association is an association between two viral proteins,that are significantly different from any protein in the mammalian hostorganism (for HSV-1, the host is humans). Although homologues of POL arepresent in mammalian cells they are considerably diverged. No cellularhomologue of UL8 is known. For the virus to overcome disruption of sucha viral protein: viral protein interaction a double mutation, i.e. amutation in each of the viral proteins involved, may be required.Alternatively the range of single mutations that overcome disruption,yet allow the two proteins to interact normally may be severelyrestricted. The probability of such reversion occurring is thusrelatively low rendering this type of interaction attractive as apotential target for therapeutic agents. Additionally, as neither UL8nor POL have close homologues in mammalian cell metabolism, the toxicityof an agent which specifically interacts with these proteins will below.

The anti-viral agent may be a peptide or more preferably a non-peptidalcompound having peptidomimetic properties. Such a non-peptidal compoundwill be preferred since it will be resistant to enzymic breakdown bypeptidases. Suitable anti-viral compounds may include peptides having anamino acid sequence derived from the C-terminal or C-proximal region ofUL8, a functional equivalent of such a peptide, or a peptidomimeticcompound therefor.

The computer program “Predict-Protein” (EMBL-Heidelberg) makes a strongprediction of the presence of an alpha-helical region near theC-terminus of HSV-1 UL8 (amino acids 709-728) with the very C-terminus(residues 729-750) predicted to be in looped or extended structures(perhaps as a “tail”). The secondary structure predictions for theC-terminal regions of the UL8 homologues of bovine herpesvirus 1(BHV-1), human cytomegalovirus (HCMV, betaherpesvirus) and Epstein-Barrvirus (EBV, gammaherpesvirus) are all similar in that an alpha-helicalregion of approximately 20 amino acids is strongly predicted to occurwithin 10-26 amino acids of the C-terminus. The most inhibitory HSV-1peptide we have identified (peptide 7, amino acids 719-738) is derivedfrom across the junction of the predicted alpha-helix and “tail”portions at the C-terminus of UL8 and is 20 amino acids in length. Weconsider it likely that the predicted conserved structures in theC-terminal regions of the other herpesvirus UL8 homologues discussedabove are similarly involved in interactions with the POL homologues andpeptides representing similar regions might be able to disrupt thePOL/UL8 interactions in these viruses. Thus the peptide is preferablyderived from the free “tail” portion and/or the α-helix portion of theC-terminus of UL8. Optionally the peptide is as small as possible, egless than 6 amino acids, but can be eg 10,14 or more amino acids inlength, particularly where the peptide is derived wholly or partiallyfrom the α-helical region of the C-terminus of UL8.

Suitable peptides are set out in Table 2. In the table of inhibitorypeptides the lower the IC₅₀ value the greater the inhibitory activity.Peptides Nos 5 and 7 are especially effective as anti-viral agents.Peptide 7 corresponding to αα 719-738 was the most inhibitory and ispreferred. Functional analogues of the peptides of Table 2 (especiallyNos 5 and 7) and peptidomimetic compounds therefor are likewise suitableanti-viral agents. Peptides derived from ααs 722-738 are particularlysuitable.

The anti-viral agent is preferably effective against a herpesvirusselected from HSV, HCMV, Human herpesvirus 8 (HHV8), EBV and EHV 1 & 4.HCMV is of particular interest. The antiviral agent is preferably alsoeffective against proteins homologous to UL8 and POL (eg UL102 and UL54respectively for HCMV). Generally the anti-viral agent will be selectedto mimic at least a portion at or near the C-terminus of the UL8homologue of the specific target virus.

In a further aspect, the present invention provides an assay todetermine the ability of a test substance to interfere with theassociation of UL8 and POL. The assay comprises the following steps:

i) providing a first viral component;

ii) exposing said first viral component to a test substance followed bya second viral component, or exposing said first viral component to asecond viral component followed by a test substance;

iii) washing to remove any second viral component and/or test substancenot associated with the first viral component; and

iv) detecting the presence, and optionally determining the amount, ofsecond viral component associated with said first viral component.

The first or second viral components may be localised on a surface, suchas a blotting membrane, or an assay plate for ELISA etc. Preferably thefirst component is immobilised in such a manner, although the inventioncontemplates the possibility of the assay being carried out in solution.

The first viral component may be POL or UL8. Where the first viralcomponent is POL, the second viral component will be UL8. Where thefirst viral component is UL8, the second viral component will be POL.

If the assay is to test the ability of the test substance to interferwith UL54/UL102 association, the first viral component may be UL54 orUL102. Where the first viral component is UL54, the second viralcomponent will be UL102. Where the first viral component is UL102, thesecond viral component will be UL54.

Detection of the presence and/or amount of second viral componentassociated with the first viral component may be conducted by anyconvenient means. Generally detection may be via a monoclonal antibody,the presence of which is established by exposure to a second labelledmonoclonal antibody in a typical ELISA-style assay. Alternatively, thesecond viral component may be labelled (eg radioactively) to determineits binding to the first viral component.

Suitable monoclonal antibodies (Mabs) for use in the assay of thepresent invention have been produced (see Examples 2 and 3) and form afurther aspect of the present invention. In particular the POL-specificMab 13185 is suitable for use in the assay of the present inventionwhere POL of HSV-1 is the second viral component. Mabs 804 and 805 areUL8-specific Mabs and are suitable for use in the present inventionwhere UL8 of HSV-1 is the second viral component. Hybridoma cell-lineshave been deposited for Mabs 13185 and 805 at the European collection ofanimal cell cultures at ECACC, Porton Down, Wiltshire on Jul. 26, 1996under Accession Nos 96072640 and 96072639 respectively.

Identification of MAb 814 as an antibody that inhibits the POL/UL8interaction and the mapping of its epitope to between amino acids 470and 671 suggests that the C-terminus may not be the only region of UL8to contribute to POL binding. For the POL/UL42 interaction theC-terminus was found to contribute 75% of the binding energy (Marsden etal 1994). The relative contribution of different regions of UL8 to POLbinding remains to be determined.

By analogy with other DNA replication systems it is considered likelythat initiation of HSV-1 DNA synthesis involves the formation of aninitiation complex at one or more of the replication origins. The firststage in this process would be the binding of UL9 to its recognitionsequence. The interaction of UL9 with UL8 might then serve to recruitthe viral helicase-primase complex (UL5, UL8 and UL52) (McLean et al.,1994). In addition, ICP8 both interacts physically with UL9 and canstimulate its helicase activity (Boehmer & Lehman, 1993; Boehmer et al.,1994). These five proteins together therefore have the potential to openup the duplex DNA in the origin region and synthesize RNA primers. Theinteraction between POL and UL8 which we have now identified may play animportant role in bringing the viral DNA polymerase (POL/UL42heterodimer) into the complex to initiate DNA synthesis. In addition adirect physical interaction between the polymerase and helicase-primasecomplexes may be important in co-ordinating the unwinding of the duplexand the synthesis of RNA primers on the lagging strand at the advancingreplication fork. This model, summarized in FIG. 10, is entirelycompatible with that proposed by Liptak et al. (1996) in which UL5, UL8,UL9, UL29(ICP8) and UL52 are assembled at prereplicative sites followedby recruitment of POL, which is facilitated by UL42. Our findingprovides the basis for the recruitment of the POL/UL42 complex. Amongstthe many questions that remain to be answered is whether the affinitiesof the different proteins for each other is influenced by the presenceof other proteins in the complex. It is possible, for example, thatbinding of POL to UL8 reduces the affinity of UL8 for UL9 allowing thehelicase-primase-polymerase complex to migrate away from the origin tothe replication forks.

The interaction of POL with UL8 may represent a possible new target forthe action of an antiviral agent. A UL8 protein lacking the C-terminal34 amino acids is unable to support viral DNA synthesis in a transienttransfection assay indicating that this region of the UL8 proteinperforms an essential replicative function. Although this providesevidence consistent with a key role for the UL8/POL interaction, itshould be noted that we cannot exclude the possibility that this regionof the protein is also necessary for some other essential function.

Our identification of peptides that block this interaction should alsoencourage further studies of this region and the search for more potentinhibitors. In the case of the HSV ribonucleotide reducase, followingthe initial discovery that peptides corresponding to the C-terminus ofthe small subunit inhibited enzyme activity (Cohen et al., 1986; Dutiaet al., 1986), it proved possible to identify more active peptidomimeticcompounds that could function intracellularly (Luizzi et al., 1994; Mosset al., 1995). The POL/UL8 interaction may be an especially attractivenew target for two reasons. First, both proteins are present in infectedcells in low amount in contrast to POL/UL42 and R1/R2 where one or bothof the interacting proteins are abundant viral products. Second, thePOL/UL8 interaction appears to be relatively weak as suggested by theobservation that in contrast to POL/UL42 and R1/R2 they do not co-purifyfrom infected cells and also by the ability of peptide 7 to block theinteraction equally effectively when pre-incubation with POL wasomitted. Such a weak interaction may be more readily blocked by anantiviral compound than a strong interaction.

Mabs 817, 818 and 819 all recognised peptide 5, that corresponds toresidues 722 to 750 of UL8, and to a lesser extent peptide 3 (aminoacids 726-750). However the Mabs do not recognise peptide 2 (amino acids728-750) or peptide 7 (amino acids 719-738). It is therefore probablethat all three MAbs recognize the same epitope located within theC-terminal 29 amino acids of UL8 and minimally involving the regionspanning amino acids 727-739.

The present invention also provides a method of combatting replicationof a herpesvirus, said method comprising providing an agent capable ofdisrupting the association UL8 and POL.

Further, the present invention provides a method of combatting aninfection caused by a herpesvirus, said method comprising administeringan antiviral agent as described above.

Additionally the present invention provides the use of an agent capableof interfering with association of POL/UL8 for combatting herpesvirusreplication or infection.

FIGURE LEGENDS

FIG. 1. Co-precipitation of POL and UL8 protein by the UL8-specificMAb804. Lanes 1 to 3 show [³⁵S]-methionine-labelled extracts from Sfcells infected with AcUL8 (lane 1), AcUL30 (lane 2) or doubly with AcUL8and AcUL30 (lane 3). The proteins precipitated from these extracts areshown in lanes 4 to 6 respectively. Proteins were separated on 8.5%SDS-polyacrylamide gels and were visualized by autoradiography. Thepositions of the POL and UL8 proteins are indicated.

FIG. 2. POL/UL8 interaction ELISAs. Panel A. UL8 protein was added tomicrotiter wells pre-coated with 0.04 μg POL (,▪) or uncoated (◯,□).Bound UL8 protein was detected with MAb804 (□,▪) or MAb805 (◯,) whichin turn were detected with an HRP-conjugated anti-mouse MAb andcalorimetric substrate. Panel B. POL was added to microtiter wellspre-coated with 0.4 μg UL8 protein (▪) or uncoated (□). Bound POL wasdetected with MAb 13185 which in turn was detected with anHRP-conjugated anti-mouse MAb and calorimetric substrate.

FIG. 3. Inhibition of the POL/UL8 interaction by UL8-specific MAbs.Ascitic fluid from UL8-specific MAbs and two control MAbs (RwP3 and105gD) were serially two-fold diluted and incubated for 1 h at 37° C.with 0.2 μg UL8 protein. The mixture was then added to microtiter wellscoated with 0.04 μg POL. After 1 h the plates were washed and bound UL8protein was detected with MAB 804 or MAb 805 as described. Theabsorbance in each well was expressed relative to that (0.945 OD units)observed in the absence of antibody. The dilutions of ascitic fluid wereas follows: ▪, 8-fold; , 16-fold; , 32-fold; , 64-fold; , 128-fold; ,256-fold; , 512-fold and □, 1024-fold. The designation of each MAb isshown below the absorbance values obtained for that MAb.

FIG. 4. Approximate mapping of the epitopes recognized by theUL8-specific MAbs. Four replicate SDS-PAGE gels were loaded with totalproteins from BHK cells lipofected with the plasmids indicated.Following electrophoresis and electroblotting of the proteins ontonitrocellulose sheets the blots were incubated with 1:2000 dilutions ofpolyclonal anti-UL8 antiserum (a), MAb 0811 (b), MAb 0814 (c) or MAb0817 (d). The blots were washed and incubated with 1:7500 dilutions ofanti-rabbit (a) or anti-mouse (b-d) IgG alkaline phosphatase conjugatedantibody and developed as described in Methods. The sizes of theprestained molecular weight markers (Sigma) are indicated.

FIG. 5. Fine mapping of the epitopes recognized by MAbs 817, 818 and819. The reactivity of the MAbs with peptides 1-8 (Table 2) was testedby ELISA. The bars show the absorbance observed with a 100-fold dilutionof each MAb. The MAbs are denoted by the different cross-hatching asfollows: , MAb 817; , MAb 818; , MAb 819.

FIG. 6. Inhibition of the POL/UL8 interaction by UL8 peptides. Differentconcentrations of peptides 5 (◯) and 7 (⋄) and the control peptide RT85(□) (Table 2) were incubated for 15 min at room temperature with 0.15 μgPOL and then added to microtiter wells coated with 0.2 μg UL8 protein.After 1 h the plates were washed and bound POL was detected withMAb13185.

FIG. 7. The sequence of amino acids in peptide 7 is important forinhibition of the POL/UL8 interaction. Different concentrations ofpeptide 7 (□) and peptide 7J (⋄), a jumbled version of peptide 7, (Table2) were incubated for 15 min at room temperature with 0.15 μg POL andthen added to microtiter wells coated with 0.2 μg UL8 protein. After 1 hthe plates were washed and bound POL was detected with MAb13185.

FIG. 8. Inhibition of the POL/UL8 interaction does not require priorincubation of peptide 7 with POL. Different concentrations of peptide 7and POL (0.15 μg) were added to microtiter wells coated with 0.2 μg UL8protein: the peptide was added either a few seconds after POL (□), orwas first pre-incubated with POL for 15 min at room temperature (⋄).After 1 h the plates were washed and bound POL was detected withMAb13185.

FIG. 9. Alignment of the sequences of peptides 7J, 7 and 5. The *indicates the positions at which amino acids in the three peptides areidentical.

FIG. 10. Interactions between the HSV-1 DNA replication proteins. Thethin, medium and thick arrows indicate relative strengths ofinteraction. In addition to binding to UL29 and UL8, UL9 also bindsspecifically to the viral replication origins. The UL9/UL29, UL9/UL8 andUL8/POL interactions are likely to be important in assembling aninitiation complex at the origins following the initial binding of UL9.

FIG. 11. Epitope mapping of POL-specific MAbs by western blotting.Extracts of E. coli cells expressing fragments 1-7 of gene UL30 encodingresidues 1-212, 162-316, 308-658, 597-975, 875-1119, 1072-1145 and1128-1235 respectively, were separated by SDS-PAGE (lanes 1-7). Lane 8contains an extract from E. coli cells transformed with the vector pQE32to serve as a control. Four such gels were blotted onto nitrocellulosemembranes and probed with polyclonal antiserum 514 (panel A), MAb 13088(panel B), MAb13129 (panel C) and MAb 13185 (panel D). The positions towhich molecular weight markers (M_(r)s 46,000, 30,000, 21,500, 14,300,6,500,and 3,400) migrate, are shown by arrows on the left of each panel.

FIG. 12. Coommassie-blue stained gel of purified proteins UL102 (lane 2)and UL54 (lane 3). Proteins were separated on an SDS-10% polyacrylamidegel. The numbers to the left of the gel show the molecular weights(×10⁻³) of the marker proteins that were electrophoresed in lane 1.

FIG. 13. Reactivity of antiserum 373, made against a peptidecorresponding to amino acids 809-823 of the predicted UL102 ORF, withextracts of Sf cells infected with recombinant virus AcUL102 andproteins at different stages of purification. Proteins were separated onan SDS-10% polyacrylamide gel, transferred to a nitrocellulose membraneand reacted with antiserum 373 (lanes 1-5) or the pre-immune serum(lanes 6-10). The electrophoresed proteins were two differentpreparations of AcUL102-infected Sf cells (EXT-1 and EXT-2, lanes 2, 3,7 and 8) together with peak UL102-containing fractions from the DEAEcolumn (DEAE-2, lanes 1 and 6) and hydroxylapatite column (HA-2, lanes 4and 9). Lanes 5 and 10 contain molecular weight markers.

FIG. 14. Specificity of antiserum 144 for UL54. ELISA wells were coatedwith 0.04 mg (,◯) or 0 μg (▾,∇) of UL54 protein and reacted withantiserum 144 (,▾) or the pre-immune serum (◯,∇) as describedpreviously (Marsden et al., 1994). The sera were initially diluted5-fold followed by serial 2-fold dilutions. Bound antibody was detectedwith HRP-conjugated protein A and colorimetric substrate.

FIG. 15. DNA-dependent DNA polymerase activity of purified UL54 protein.Incorporation of [³H]dTTP into an poly(dA)-oligo(dT)₁₂₋₁₈ template by 10ng protein () or no protein (▪).

FIG. 16. The HSV-1 UL30/POL interaction can be detected by rabbitantibody 113 that was raised against the C-terminal 15 amino acids ofHSV-1 UL30. The data is presented as 6 groups (A-F) each comprising 4absorbance values (1-4). The absorbance values represent data from theinteraction assay as follows: 1, both UL30 and UL8 proteins present; 2,UL8 only; 3, UL30 only; 4, both UL30 and UL8 proteins absent. The groupscorrespond to absorbance values produced with detecting antibodies asfollows: A, MAb 13815 diluted 1/50; B, no monoclonal antibody; C-E,Rabbit antiserum 113 diluted 1/10³, 1/10⁴, 1/10⁵ respectively; F norabbit antiserum. The presence of bound antibody was detected withHRP-conjugated goat anti-mouse antibody (groups A and B) orHRP-conjugated protein A (groups C-E) and calorimetric substrate.

FIG. 17. UL54/UL102 interaction ELISA. UL54 protein was added tomicrotiter wells pre-coated with UL102 protein. The amounts of UL102protein used to coat the wells were as follows: 0.4 μg (), 0.2 μg (◯),0.1 μg (▾), 0.02 μg (∇), or uncoated (▪). Bound UL54 was detected withrabbit antiserum 114 which in turn was detected with HRP-conjugatedprotein A and calorimetric substrate.

FIG. 18. Inhibition of the UL54/UL102 interaction by UL102 peptides.Different concentrations of peptides 1 () and 2 (◯) and the controlpeptide RT85 (▾) (Table 4) were added with 0.4 μg UL54 to microtiterwells coated with 0.4 μg UL102 protein. After 1 h the plates were washedand bound UL54 was detected with HRP-conjugated protein A andcolorimetric substrate.

SEQUENCE LISTINGS SEQ ID NO 1 Peptide 1 (Table 2) SEQ ID NO 2 Peptide 2(Table 2) SEQ ID NO 3 Peptide 3 (Table 2) SEQ ID NO 4 Peptide 4 (Table2) SEQ ID NO 5 Peptide 5 (Table 2) SEQ ID NO 6 Peptide 6 (Table 2) SEQID NO 7 Peptide 7 (Table 2) SEQ ID NO 8 Peptide 8 (Table 2) SEQ ID NO 9Peptide 7J (Table 2) SEQ ID NO 10 Peptide RT85 (Table 2) SEQ ID NO 11Peptide 1 (Table 4) SEQ ID NO 12 Peptide 2 (Table 4) SEQ ID NO 13C-terminal 15 amino acids of UL54 SEQ ID NO 14 Residues 809-823 of the873 residue UL102 SEQ ID NO 15 Primer (Example 3) SEQ ID NO 16 Primer(Example 3) SEQ ID NO 17 Primer (Example 3) SEQ ID NO 18 Primer (Example3)

The present invention will now be further described with reference tothe following, non-limiting, examples.

EXAMPLE 1

POL/UL8 Association

Materials and Methods

Cells and recombinant baculoviruses. Spodoptera frugiperda (Sf) cells(strain IPLB-SF-21; Kitts et al., 1990) were maintained in TC100 medium(Life Technologies) containing 5% (v/v) fetal calf serum (FCS),penicillin (100 units/ml) and streptomycin (100 μg/ml). The Autographacalifornica nuclear polyhedrosis virus (AcNPV) recombinants AcUL30,AcUL8 (which overexpress POL and UL8 proteins respectively) have beendescribed (Stow, 1992; 1993). Preparation and titration of virus stockswere carried out as described (Brown and Faulkner, 1977; Matsuura etal., 1987).

Antibodies. The isolation of one UL8-specific monoclonal antibody (MAb)following immunisation of mice with purified UL8 protein was describedpreviously (McLean et al., 1994). An additional 19 MAbs were isolatedfrom the same fusion and screened for reactivity with UL8 protein byELISA. Ascitic fluid was prepared from cells secreting UL8-reactiveantibodies. Two control MAbs were also used in these experiments: MAbRwP3 is secreted from the P3-X67-Ag8 myeloma cells (Kohler andMilstein,1975) and MAb105 gD reacts with glycoprotein D of HSV-1 (A.Cross, unpublished data). Polyclonal rabbit antiserum 094 was preparedin a Sandy Half-Lop rabbit which was immunized intramuscularly, firstwith approximately 25 μg purified UL8 protein (Parry et al., 1993) inFreund's complete adjuvant, followed by three boosts 10, 30 and 40 dayslater using the same amount of antigen but in Freund's incompleteadjuvant. The animal was sacrificed on day 50 and serum was collected.

Immunoprecipitation, immunofluorescence and Western blotting. Theprocedures used to prepare [³⁵S]-methionine labelled extracts from Sfcells infected with recombinant baculoviruses have been described indetail (McLean et al., 1994). The extracts were incubated with 1.0 μl ofascitic fluid of UL8-specific MAb804, immune complexes were captured onProtein A-Sepharose beads, proteins were separated on 8.5% SDSpolyacrylamide gels and were then visualized by autoradiography asdescribed in detail previously (McLean et al., 1994). Immunofluorescenceand Western blotting were performed as described (Calder et al., 1992;McLean et al., 1994). Briefly, Sf cells infected with AcUL8 wereharvested 2 days after infection, washed with PBS and solubilised withdenaturing sample buffer (Laemmli, 1970), separated by 10% SDS-PAGE, andtransferred to nitrocellulose membranes. The membranes were incubatedwith MAbs diluted from 10²- to 10⁵-fold, and bound antibodies werevisualised using HRP coupled to anti-mouse Ig (Sigma), and chromogenicsubstrate 4-chloro-1-naphthol (Bio-Rad).

POL/UL8 interaction assays. ELISA assays, similar to that described forPOL/UL42 (Marsden et al., 1994) were established with purified POL andUL8 proteins. POL was extracted from Sf cells infected with recombinantbaculovirus AcUL30 and purified as described by Gottlieb et al. (1990)with minor modifications (Marsden et al., 1994). UL8 protein wasextracted from Sf cells infected with recombinant baculovirus AcUL8 andpurified as described by Parry et al., (1993) but substitutingphenyl-Sepharose by hydroxylapatite chromatography (Dodson and Lehman,1991). Both proteins were diluted in PBS to the required concentrations.For the first assay, microtiter wells were coated overnight with 0.02 μgof purified POL and blocked with 100 μl of 2% BSA in PBS for 1 h at 37°C. After blocking, the plates were washed extensively with PBScontaining 0.3% Tween 20 and blotted dry. Then 50 μl of purified UL8, atthe concentrations indicated in the text, were added to each well andincubated for 1 h at 37° C. Following further washes, 50 μl ofUL8-specific MAb 804 or MAb 805 diluted 1:400 in PBS containing 2% FCSwas reacted for 1 h at 37° C. The wells were again extensively washedand bound MAb was detected with 50 μl/well of HRP-conjugated goatanti-mouse IgG (Sigma) diluted 1:500 in PBS containing 2% FCS andchromogenic substrate ABTS. For the second assay, POL and UL8 proteinswere added in the reverse order. Microtitre wells were coated overnightwith 0.02 to 0.04 μg of purified UL8 protein and bound POL was detectedwith a POL-specific MAB 13185 (Marsden et al., 1996) diluted 1:100.Other aspects of the two assays were identical. MAbs, diluted in PBSplus 2% fetal calf serum, and peptides, diluted in 100 mM Tris-HCl (pH8.0) plus 0.1% Tween 20, were added to the interaction assay asdescribed in the text.

Oligopeptides. Peptides (Table 2) were synthesized by continuous flowFmoc chemistry as previously described (Atherton and Sheppard, 1989;McLean et al., 1991). Peptides were purified by preparativereverse-phase HPLC (Owsianka et al., 1993). The Mr values of monomericpeptides were determined by matrix-assisted, laser desorptiontime-of-flight (MALDI-TOF) mass spectrometry and corresponded to thedesired products.

Expression of fragments of UL8 and mapping UL8-specific MAbs. PlasmidpE8 contains the UL8 DNA replication gene under the control of the humancytomegalovirus major immediate early promoter in the vector pCMV10(Stow et al., 1993) and served as parent for the construction ofplasmids expressing N- and C-terminally truncated UL8 proteins,designated pNΔx and pCΔx, where x corresponds to the number of aminoacids deleted (E. C. Barnard and N. D. Stow, manuscript in preparation).BHK cells (approximately 1.5×105 per 35 mm diameter petri dish) weretransfected with 2 μg wild type pE8 or deletion mutant DNA usingliposomes prepared as described by Rose et al. (1991). 30 hpost-transfection the cells were washed with PBS and total cell proteinsprepared by treating the monolayers with 150 μl denaturing sample buffer(Laemmli, 1970). Protein samples (usually 20 μl) were subjected toelectrophoresis through 9% polyacrylamide gels using a Bio-Rad miniprotein gel kit and electroblotted onto a nitrocellulose membrane(Towbin et al., 1979). Replicate membranes were reacted with a 1 in 2000dilution of the MAb or with a 1 in 2000 dilution of rabbit polyclonalantiserum 094 against whole UL8 protein, and bound antibody was detectedusing alkaline phosphatase-conjugated anti-mouse or anti-rabbit IgGsecondary antibody, as appropriate, in conjunction with the PromegaProtoblot system.

Fine mapping of MAbs with peptide-based ELISA assays. Peptides werediluted in 100 mM Tris-HCl (pH 8.0) plus 0.1% Tween 20 and coatedovernight onto microtiter wells at 1.0 μg/well in 50 μl. The wells werethen blocked and washed as described above. MAbs were diluted 100-foldin PBS plus 2% FCS and 50 μl was added to each well and incubated for 1h. The antibody was removed, plates were again washed and bound antibodywas determined with HRP-conjugated anti-mouse IgG and colorimetricsubstrate as described above.

Results

Isolation and characterisation of MAbs reactive with UL8 protein. Theisolation of a single MAb, (designated 0811) following immunisation ofmice with purified UL8 has been described (McLean et al., 1994). Fromthe same fusion a further 19 cell lines secreting MAbs reactive with UL8protein were isolated. Ascitic fluid was developed for each cell lineand screened for reactivity with UL8 protein in immunoprecipitation,immunofluorescence and Western blotting assays. Four of the MAbs werefound to react strongly by Western blotting with protein(s) fromuninfected BHK cells and were not studied further. The results obtainedfor the remaining 16 MAbs are summarized in Table 1.

Co-precipitation of POL with OL8. MAbs capable of immunoprecipitatingUL8 protein have also been examined for their ability to co-precipitateother viral DNA replication proteins from [³⁵S]-methionine-labelledextracts of Sf cells mixedly infected with recombinant baculoviruses. Wepreviously described the identification of an interaction between UL8and UL9 using this approach (McLean et al., 1994). When extracts from Sfcells co-infected with AcUL8 and AcUL30 were reacted with MAb804, POLwas found to co-precipitate with UL8 (FIG. 1, lane 6). Precipitation ofPOL was specific and dependent on the presence of UL8 protein since noprotein of a similar size was detected in the immunoprecipitates fromextracts of cells infected singly with either AcUL8 (lane 4) or AcUL30(lane 5).

ELISAs to measure the POL/UL8 interaction. To investigate theinteraction between POL and UL8 in greater detail, two separate ELISAswere developed. In one, POL was coated onto microtiter wells and bindingof added UL8 was monitored with a UL8-specific MAb, while in the otherassay, UL8 was coated onto microtiter wells and binding of added POL wasmonitored with a POL-specific MAb. The purified proteins used for theassay were essentially homogeneous as judged by SDS-PAGE analysis andcoomassie blue staining: representative preparations have been shown inearlier publications (Marsden et al., 1994; Parry et al., 1993). In thefirst assay, binding of UL8 protein to POL-coated microtiter wells wasdetected with either MAb804 or MAb805 and the absorbance was dependenton the presence of the antibody (data not shown). FIG. 2A shows thecharacteristics of the assay and demonstrates that the amount of eitherMAb bound was dependent on the presence of both POL and UL8 proteins. Anamount of 0.04 μg of POL was sufficient to give a good signal in thisassay and was used throughout. At amounts of UL8 above 0.2 μg/well someabsorbance was detected in the absence of POL. Therefore, 0.2 μg of UL8protein per well was used in all subsequent experiments which producedan absorbance that corresponded to nearly the top of the steep initialrise.

In the second assay, the most sensitive of the POL-specific antibodies(Marsden et al., 1996) that were tested for detection of POL-binding toUL8-coated wells was MAb 13185 (FIG. 2B) and this MAb was usedthroughout subsequent experiments. Other POL-specific antibodies, eg.MAb 13088 and MAb 132129, gave about half the signal, while MAb 13429and a control MAb, RwP3, gave no signal (data not shown). Again thesignal was dependent on the presence of both POL and UL8 proteins (FIG.2B) and POL-specific antibody (data not shown). Preparations of purifiedPOL and UL8 protein were again titrated to determine the optimum amountsto be used in this assay. It was found that 0.2 to 0.4 μg/well of UL8and 0.15 to 0.2 μg of added POL gave a good signal corresponding tonearly the top of the steep initial rise. These two ELISAs thus providefast and convenient assays for monitoring the interaction between thetwo proteins.

Specific inhibition of the POL/UL8 interaction by UL8-reactive MAbs.Since it was possible that some of the MAbs reactive against UL8 mightbind the molecule close to the site of interaction with POL, theUL8-specific MAbs were screened for ability to inhibit UL8 binding toPOL-coated microtiter wells. Two MAbs, RwP3 and 105gD, that did notreact with UL8 protein were used as controls. Doubling dilutions of eachascitic fluid were made, starting with an 8-fold dilution, and weremixed with an equal volume of UL8 protein to give a final concentrationof 0.2 μg of UL8 protein per 50 μl. After incubation for 1 h at 37° C.,the mixture was added to POL-coated microtiter plates and the assay wasprocessed in the usual manner. The average absorbance in 14 wells in theabsence of any MAb was 0.945 (standard deviation=0.122) and allabsorbance readings were normalized to this value so that a relativeabsorbance of 1 corresponds to an absorbance of 0.945. The results for13 of the UL8-specific MAbs are presented in FIG. 3 in which therelative absorbance values for each of the 8 concentrations tested foreach MAb are presented as bars: the filled bar represents the 8-folddilution and subsequent doubling dilutions are represented byprogressively less densely shaded bars until the 1024-fold dilution openbar. Three patterns or reactivity were observed. The top panel containsthose antibodies that did not reduce the relative absorbance below 0.50and which were classified as non-inhibitory. The middle panel containsthose antibodies that reduced the relative absorbance to less than 0.25and were classified as inhibitory. The bottom panel contains antibodiesthat reduced the absorbance to between 0.25 and 0.50. This latter groupof antibodies was not classified. Each antibody was tested between 2 and4 times and the results from experiment to experiment were in goodagreement. The behaviour all 16 MAbs is summarized in Table 1, whichlist the 5 consistently inhibitory antibodies. The epitopes recognizedby those antibodies that inhibit the POL/UL8 interaction are likely tolie at or near residues on UL8 involved in its interaction with POL.

Mapping of the epitopes recognized by the UL8-specific MAbs. Seven ofthe 8 MAbs which detected insect cell-expressed UL8 in a Western blotwere also sufficiently sensitive to allow detection of UL8 expressed inBHK cells transfected with plasmid pE8 (MAbs 809, 811, 812, 814, 817,818 and 819). In order to determine approximate locations for theepitopes recognised, we tested their ability to detect a series of N-and C-terminally truncated UL8 molecules expressed from derivatives ofpE8. Western blots, each containing an identical array of extracts fromBHK cells transfected with mutated plasmids, were reacted withindividual MAbs or with a polyclonal anti-UL8 antiserum (094) andrepresentative results are shown in FIG. 4. The polyclonal serumefficiently detected each of the UL8 products (panel A). Threedistinctive patterns of reactivity were observed with the MAbsindicating that the epitopes mapped to three distinct regions. MAbs 811(B) and 812 (not shown) reacted with all the truncated proteinsindicating that they recognized an epitope lying in the region of aminoacids 165-253 (designated as region 1). MAbs 809 (C) and 814 (not shown)reacted with all the N-terminally truncated proteins and with productslacking up to 79 but not 280 amino acids from their C-terminusindicating the presence of an epitope between amino acids 470 and 671(region 2). Further analysis revealed that both these MAbs were able todetect a deleted form of UL8 lacking 196 amino acids from its C-terminusthereby narrowing down the region containing the epitope(s) to aminoacids 470-554 (data not shown). It is not yet known whether the MAbsrecognized the same or distinct epitopes within regions 1 and 2.

MAbs 817 (D), 818 and 819 (not shown) failed to react with truncatedproteins lacking 33 or more amino acids from their C-termini suggestingthat they recognize one or more epitopes close to the C-terminus of UL8(amino acids 717-750, region 3). To determine whether amino acids fromthis region were sufficient for recognition and to define the epitope(s)more closely series of 8 overlapping peptides that spanned and extended3 amino acids upstream of region 3 were synthesized (Table 2) and testedfor reactivity by ELISA with the MAbs. FIG. 5 shows that the three MAbsbehaved identically and reacted predominantly with the C-terminal 29amino acids of UL8 contained in peptide 5 and to a lesser extent withthe slightly shorter peptides 3 and 4. Removal of 4 amino acids from theN-terminus or 12 from the C-terminus of peptide 5 reduced the signal tobackground levels. It is therefore probable that all three MAbsrecognize the same epitope located within the C-terminal 29 amino acidsof UL8 and minimally involving the region spanning amino acids 727-739.

Inhibition of the POL/UL8 interaction by UL8 peptides. The finding thatall three of the MAbs (817, 818 and 819) that mapped within theC-terminal 29 amino acids of UL8 inhibited the interaction between UL8and POL prompted us to examine the role of the C-terminal amino acids ofUL8 in binding. Peptides 1-8 (Table 2) were tested for their ability toblock the interaction of UL8 with POL. Peptides were dissolved in 100 mMTris-HCl (pH 8.0) plus 0.1% Tween 20 at different concentrations andincubated with POL for 15 min to allow the peptides to bind to POL. Themixtures were then added to microtiter wells coated with UL8 and theamount of POL bound was determined after 1 h. FIG. 6 shows the resultsfor peptides 5 and 7 and a control peptide, RT85. Peptides 5 and 7,which could not be analysed at concentrations higher than those shownbecause of their limited solubility, were markedly inhibitory. Thecontrol peptide was non-inhibitory, even at 500 μM. The concentration ofeach peptide required to reduce POL binding by 50% (the IC₅₀ value) wasdetermined in at least 3, and on average 5, independent experiments. Theaverages of these values are listed in Table 2 together with thestandard deviations for peptides 5 and 7. The different IC₅₀ values,ranging from 2.25 μM to non-inhibitory, suggest that the observedinhibition is peptide-specific.

To obtain additional evidence for sequence specificity of the mostinhibitory peptide, an additional peptide was made that contained thesame amino acids as peptide 7 but in jumbled order, and was tested forinhibition of the POL/UL8 interaction. The jumbled peptide, 7J, was madeby linking residues from alternately the N- and C-termini of peptide 7.Thus, if the order of the 20 residues in peptide 7 is represented as 1,2, 3, . . . 18, 19, 20, that in 7J was 1, 20, 2, 19, . . . 12, 10, 11.In the experiment shown in FIG. 7, the IC₅₀ value for peptide 7 wasapproximately 1 μM while peptide 7J was 20-fold less active with an theIC₅₀ value >20 μM.

Inhibition of the POL/UL8 interaction does not require prior incubationof peptide 7 with POL. In the previous experiment, the peptides had beenpre-incubated with POL to increase the likelihood that they wouldinhibit its interaction with UL8 by allowing prior formation of apeptide-POL complex. We next investigated whether the preincubation stepwas necessary. The data presented in FIG. 8 show that the IC₅₀ values(approximately 1 μM) for the two curves are indistinguishable,demonstrating that inhibition does not require prior incubation with thepeptide and suggesting that the POL/UL8 interaction might be a weak one.

EXAMPLE 2

Isolation and Characterization of POL-specific Monoclonal Antibodies

Materials and Methods

Cells. P3-X67-Ag8 myeloma (P3) cells were grown in Dulbecco's MEM with10% foetal calf serum, 10% horse serum, 8 mM glutamine and gentamicin.Spodoptera frugiperda (Sf) cells were grown at 28° C. in TC100 mediumwith 5% heat-inactivated foetal calf serum, antibiotics and neomycin.All reagents were supplied by Gibco/BRL and used as recommended by thesuppliers except where noted.

Production and purification of proteins. POL was extracted (Gottlieb etal., 1990) from Sf cells infected with recombinant baculoviruses AcUL30(Stow, 1992). POL protein was purified by a modification (Marsden etal., 1994) of the procedure described by Gottlieb et al. (1990).

DNA fragments encoding portions of POL were subcloned from plasmid pE30which encodes the full length protein (Stow et al, 1993). Convenientrestriction endonuclease fragments of the UL30 gene were purified andinserted in-frame into the appropriate vector from the pQE30, pQE31,pQE32 series (Qiagen). The resulting plasmids specify fusion proteinswith an N-terminal extension of approximately 25 αα, including a stretchof 6 histidine residues. E.coli strain XL-1 blue cells (Stratagene)transformed with the following plasmids were used:—pPQ223 encoding αα1-212 (fragment 1), pPQ101 encoding αα 162-316 (fragment 2), pPQ3encoding αα 308-658 (fragment 3), pPQ117 encoding αα 597-975 (fragment4), pPQ24 encoding αα 875-1119 (fragment 5), pPQ136 encoding αα1072-1145 (fragment 6) and pPQ131 encoding αα 1128-1235 (fragment 7).XL-1 blue cells transformed with the vector pQE32 served as a control.Synthesis of the UL30 fragments was induced following treatment ofE.coli cultures with IPTG (optimum conditions were 0.1-1.0 mM IPTG for1-5 hours depending on the construct).

Preparation of antibodies. Donor mice for MAb production received 3subcutaneous injections at weekly intervals, the first with completeFreund's adjuvant (CFA) and the next two with incomplete Freund'sadjuvant (IFA). Three to five weeks later the mice were boosted withantigen in PBS intraperitoneally and test bled. Spleen donors received afurther boost with antigen intraperitoneally four days before thefusion. Mouse spleen cells were fused to SP3 cells in the UL30 fusionusing polyethylene glycol 1000. Fused cells were plated at 3×10⁵cells/well in selective medium containing HAT. Purified UL30 protein wasused both for immunising donor mice and for assaying secreted antibodiesby ELISA. The amount of protein for each of the 3 initial immunisationswas 10 μg for POL, while for the boosts 20 μg of POL were used. ELISAassays for mouse antibodies were performed with the spent medium ofgrowing hybridoma cells or with immunoglobulin (Ig) purified from it.Purification was achieved by precipitation of the Ig with ammoniumsulphate at 50% saturation or by a more rigorous procedure whereby themedium was dilapidated with Cab-o-sil (BDH) followed by ammoniumsulphate precipitation. Finally, the precipitate was dissolved in anddialysed against 20 mM Na-phosphate buffer pH7.0, and further purifiedusing a protein G suberose column (Pharmacia): bound Ig was eluted with0.1M glycine pH2.7, neutralised, aliquoted and stored frozen.

Polyclonal antisera 514 specific for POL was raised in rabbits by 5intramuscular injections at fortnightly intervals, each of 5 μg ofpurified POL. The first immunisation was in CFA while subsequentimmunisations were in IFA.

ELISA. Plates were coated at 37° C. overnight with 0.25 μg per well ofpurified UL30 protein: an amount chosen by initial checkerboardtitrations. Tissue culture supernatants were added for 1 hour at ambienttemperature. Bound antibodies were detected with horse radish peroxidase(HRP) coupled to anti-mouse Ig (Scottish Antibody Production Unit), andthe chromogenic substrate ABTS (Sigma).

Western blots. Cells were solubilised with denaturing sample buffer andproteins were separated by 17.5% SDS polyacrylamide gel electrophoresis(SDS-PAGE) and transferred to nitrocellulose membranes. The membraneswere incubated with purified Ig, and bound Ig was visualised using HRPcoupled to anti-mouse Ig for Mabs or protein A for rabbit antibodies(both Sigma), and chromogenic substrate 4-chloro-1-naphthol (Bio-Rad).Molecular weights were estimated by comparison with standard markers(Amersham 46K-2.35K).

Immunoprecipitation. Sf cells were infected with recombinant virusAcUL30 (Stow, 1992) or parental virus AcRP23lacZ (Possie & Howard,1987), and incubated at 28° C. overnight. Infected cells were labelledwith 100 uCi/ml of [³⁵S] methionine (Amersham) from 24 to 31 hourspost-infection (pi). They were then washed and solubilised in extractionbuffer (0.5% Nonidet P40, 0.5% Na deoxycholate, 10% glycerol, 0.1M TrisHCl, pH8) for 1 hour on ice. 50μl hybridoma supernatant was incubatedovernight at 4° C. with 20 μl of ³⁵S-labelled extract and 5 μl sheepanti-mouse Ig. Complexes were precipitated with protein A Sepharose,eluted by boiling with elution buffer (2% SDS, 5% 2-mercaptoethanol, 20%glycerol, 0.125M tris HCl, pH6.8, bromphenol blue) and separated by5-12.5% gradient SDS-PAGE.

Immunofluorescence. BHK cells grown on coverslips were infected withapproximately 5 plaque forming units wt HSV-1 per cell. At 5 hour picells were fixed in 2% paraformaldehyde and permeabilised with 0.5%Nonidet P40. Coverslips were incubated first with the monoclonalantibody (undiluted supernatant) and then incubated with anti-mouse Igconjugated to fluorescein isothiocyanate and examined under a Nikonmicrophot-SA microscope.

Results

Isolation and characterisation of POL-specific Mobs.

Fourteen hybridoma lines were developed that secreted antibodies whichbound specifically to purified POL coated onto microtiter plates. Theseantibodies were tested for reactivity in immunoprecipitation,immunofluorescence and western blotting assays and the findings aresummarised in Table 3. Eight of the 14 MAbs were positive inimmunoprecipitation assays and reacted with a single major protein ofthe size expected for intact POL (data not shown). Of the four MAbs thatwere positive in immunofluorescence assays, the most strongly reactivewas 13429. Eight MAbs were positive by western blotting. Three MAbs,(13185, 13429 and 13628) were reactive in all of the immunologicalassays.

The epitopes on POL recognised by those MAbs that reacted on westernblots, were mapped using a series of seven fragments of gene UL30 thatspanned the entire open reading frame. The fragments, designated 1 to 7,contained POL residues 1-212, 162-316, 308-658, 597-975, 875-1119,1072-1145 and 1128-1235 and have expected molecular masses of 23200,17822, 38858, 41863, 27030, 7718 and 11581 respectively. With theexception of fragment 1 with which no reactivity was observed, thePOL-specific polyclonal antiserum 514 reacted with a polypeptidecompatible (within the limits of SDS-PAGE) with the expected size ofeach of the fragments (FIG. 11) demonstrating the presence of fragments2-7 in the extracts. The faster migrating bands reactive with antiserum514 are probably proteolytic breakdown products of the fragments. PanelsB, C and D show the specific recognition of fragments 3, 2 and 5 by MAb13088, MAb 13129 and MAb 13185 respectively. The fragments recognised bythe other western-blot reactive MAbs were determined in the same manner(data not shown) and all are listed in Table 3, together with thededuced approximate location of the epitope.

Discussion

To our knowledge, this is the first isolation and characterisation ofmonoclonal antibodies specific for the POL protein of HSV. Within thefourteen MAbs specific for the catalytic subunit of the DNA polymerase,eight distinct specificities can be recognised. This can be deduced fromthe data in Table 3, which shows that six distinct patterns ofreactivities (A-F) in immunofluorescence (IF), immunoprecipitation (IP)and western blotting (WB) assays were observed. Pattern A, representedby MAbs 13185, 13429 and 13628, is formed by those MAbs that arepositive in all three assays. It contains two specificities: MAbs 13185and 13429 recognise an epitope between αα 976-1071 whereas the epitoperecognised by MAb 13628 lies between αα 1120-1127. Similarly, MAb 13129,13488 and 13528, comprising pattern B, define two epitopes.

None of the POL-specific antibodies inhibited DNA polymerase activity.The nine different epitopes recognised by the antibodies are widelyspread over POL though none is located near its C-terminus. Thesefindings are consistent with our earlier observation that the C-terminal27 αα of POL are responsible for at least 75% of the binding energy ofPOL to UL42 protein. Interestingly, of the panel of 13 UL42-specificMAbs that was recently described by Scheaffer et al. (1995), all but oneof the epitopes were outside the minimal active portion of the proteinand none interfered with the POL/UL42 interaction.

EXAMPLE 3

Demonstration of an Interaction Between HCMV UL54 and UL102 Proteins andInhibition of that Interaction by UL102 C-terminal and C-proximalPeptides

Methods

Expression of the HCMV UL102 and UL54 gene products. Recombinantbaculoviruses expressing HCMV genes UL102 and UL54 under the control ofthe polyhedrin promoter were generated as described below.

A 3.7 kb fragment spanning nucleotides 76904-80636 of HCMV DNA (Chee etal., 1994) and containing the HCMV UL54 ORF was amplified from a clonedcopy of the HindIII F fragment of HCMV strain AD169 by PCR. The primersused were: 5′-ATTATCTAGACCGCTATGTTTTTCAACCCG-3′ and5′-TATATCTAGACATCATCACCGTCCCCAGTCA-3′ which contained XbaI sites(underlined). The PCR-generated fragment was cleaved with XbaI andinitially cloned into the XbaI site of pUC19. The XbaI fragment was thenrecloned into the XbaI site of the baculovirus transfer vector pAcYMX1(Stow, 1992) downstream of the polyhedrin promoter to generate plasmidPY54. The entire XbaI fragment was sequenced to confirm the presence ofthe authentic UL54 gene.

A 2.7 kb fragment spanning nucleotides 146510-149208 of HCMV DNA (Cheeet al., 1994) and containing the HCMV UL102 ORF was amplified from acloned copy of the HindIII R fragment of HCMV strain AD169 by PCR. Theprimers used were 5′-ATTA GGATCCTTCTGTCCGAGGATGACCGCT-3′ and5′-ATTAGGATCCACGTCACACGCTAAGAGC-3′ which contained BamHI sites(underlined). The PCR-generated fragment was cleaved with BamHI andcloned firstly into the pUC19 BamHI site. The UL102-containing BamHIfragment was then inserted into the BamHI site of transfer vector pAcYMl(Matsuura et al., 1987) to generate plasmid PY102. The presence of theauthentic UL102 gene was confirmed by DNA sequencing of the entire BamHIfragment.

The transfer plasmids (PY54 and PY102) were separately cotransfectedwith Bsu36I-cleaved DNA of the parental baculovirus AcPAK6 (Bishop,1992) into Spodoptera frugiperda (Sf) cells and recombinantbaculoviruses were isolated as described by Kitts et al. (1990). Thepresence of the desired genes was confirmed by Southern blot analysisusing the amplified fragments as probes. Resulting viruses AcUL54 andAcUL102 contain the UL54 and UL102 genes, and stocks were prepared andtitrated as described (Brown and Faulkner, 1977; Matsuura et al., 1987).

Purification of UL54 and UL102. Proteins UL54 and UL102 were extractedfrom Sf cells infected with recombinant baculoviruses AcUL54 and AcUL102and purified as was described for the HSV-1 homologues UL30 and UL8respectively (see POL/US interaction assays, Example 1).

Measurement of DNA polymerase activity. Activity was measured byincorporation of [³H]dTTP into a poly(dA)-oligo(dT)₁₂₋₁₈ template usinga concentration of 50 mM KCl, previously found to be optimal for theHCMV enzyme (Ertl et al., 1991). The reaction mixture (final vol. 100μl) contained 75 mM Tris HCl pH 8.0, 1.67 mM 2-mercaptoethanol, 6.5 mMMgCl₂, 1 μg poly (dA)-oligo (dT), 50 mM KCl, 40 μg BSA and 10 ng UL54protein (HCMV DNA polymerase). Reactants were mixed on ice and thereaction was initiated by addition of 1.7 μM ³H-dTTP (specific activity3.75 Ci/mmol) and transfer to 37° C. Samples of 10 μl were taken 5, 10,15 and 20 minutes later, and spotted onto Whatman DE81 ion exchangediscs which had been soaked in 0.1M EDTA and air dried. The discs weregiven three 10 minute washes with 5% Na₂HPO₄, two 5 minute washes withwater and two 30 second washes with methylated spirits. They were airdried and counted in a scintillation counter with 5 ml of Ecoscint A(National Diagnostics, Kimberley Research).

Oligopeptides. Peptides were synthesized by continuous flow Fmocchemistry as previously described (Atherton and Sheppard, 1989; McLeanet al., 1991). The peptides listed in Table 4 were purified bypreparative reverse-phase HPLC. The relative molecular masses of thepurified peptides was determined by matrix-assisted, laser desorptiontime-of-flight mass spectrometry and corresponded to the desiredproducts.

Antibodies. The hybridoma cell line that secretes monoclonal antibody(MAb) 13815 has been deposited with the European Collection of CellCultures (reference number 96072640). Antiserum 113, specific for HSV-1UL30 (POL), was raised against a peptide corresponding to the C-terminal15 amino acids of the protein and has been described previously (Marsdenet al., 1994). Antiserum 144, specific for HCMV UL54 protein, was raisedin rabbits-against peptide HLEPAFLPYSVKAHE that corresponds to theC-terminal 15 amino acids (residues 1226-1240) of UL54. Antiserum 373,specific for HCMV UL102 protein, was raised in rabbits against peptideVLSSALPSVTSSSSG that corresponds to residues 809-823 of the 873 residueUL102. The peptides were made as multiply antigenic peptides (Tam, 1988)of general structure (peptide sequence)₄K₃A as such peptides have beenshown to generate sera with higher anti-protein titers (McLean et al.,1991).

UL54/UL102 interaction assays. ELISA assays, similar to those describedfor HSV-1 POL/UL8 (see Example 1 above) were established with purifiedHCMV UL54 and UL102 proteins. Both proteins were diluted in PBS to therequired concentrations. For the assay, microtiter wells were coatedovernight with purified UL102, at the concentrations indicated in thetext, and blocked with 100 μl of 2% BSA in PBS for 1 h at 37° C. Afterblocking, the plates were washed extensively with PBS containing 0.3%Tween 20 and blotted dry. Then 50 μl of purified UL54, at theconcentrations indicated in the text, were added to each well andincubated for 1 h at 37° C. Following further washes, 50 μl ofUL54-specific antiserum, diluted in PBS containing 2% FCS was reactedfor 1 h at 37° C. The wells were again extensively washed and boundantibody was detected with 50 μl/well of HRP-conjugated protein A(Sigma) diluted 1:500 in PBS containing 2% FCS. After further washes,chromogenic substrate ABTS was added. Peptides, diluted in 100 mMTris-HCl (pH 8.0) plus 0.1% Tween 20, were added to the interactionassay as described in the text.

Results

Nucleotide sequence of HCMV gene UL102. Our independently determinedsequence of the entire cloned fragment that spanned nucleotides146510-149208 (data not shown) was the same as that originally reportedin the sequence of the entire genome of HCMV strain AD169 (Chee et al.,1994) with the exception of nucleotide 146753. In agreement with Smithand Pari (1995), we found that this residue is a cytosine rather than aguanosine that changes the putative in-frame stop codon TAG at position146751 to the tryptophan codon TAC. We therefore concur with theinterpretation of Smith and Pari (1995), that the first in-frame stopcodon is at nucleotide 149105 and that gene UL102 has the capacity toencode a protein of 873 amino acids with a molecular mass ofapproximately 100K.

Purification of proteins. HCMV UL102 and UL54 proteins were extracted inthe same buffers, and purified by the same procedures previously usedfor the homologous HSV-1 UL8 and UL30 proteins (see above). Purificationwas monitored using the UL102- and UL54-specific antisera 373 and 144respectively. FIG. 12 shows a Commassie blue stained gel of purifiedUL102 (lane 2) and UL54 (lane 3). The marker proteins show that theproteins migrate to positions compatible with their predicted sizes.

Additional evidence for the authenticity of the UL102 protein wasprovided by the specific reaction of immune serum 373, but not thepre-immune serum, with the protein. FIG. 13 shows a western blot of twodifferent extracts from AcUL102-infected Sf cells (EXT-1 and EXT-2)together with peak UL102-containing fractions from the DEAE-sepharosecolumn (DEAE-2) and the hydroxylapatite column (HA-2). The purificationprocedure removes a number of faster migrating UL102-related proteinbands. It is noteworthy that the pre-immune serum does not react withany bands in these same fractions (Lanes 6-9). Alignment of the blotwith the Commassie blue stained gel showed that the band that reactedwith antibody 144 comigrated with the purified protein and migrated tothe same position with respect to the protein markers (alignment notshown).

Additional evidence for the authenticity of the UL54 protein wasprovided by the specific reaction of immune serum 144, but not thepre-immune serum, with the protein (FIG. 14). Furthermore, the purifiedprotein was able to catalyse incorporation of [³H]dTTP into anpoly(dA)-oligo(dT)₁₂₋₁₈ template as would be expected of the catalyticsubunit of the HCMV DNA polymerase (FIG. 15).

Development of an ELISA to measure the UL54/UL102 interaction. Toprovide evidence that the UL54-specific antiserum 144, directed againstthe C-terminal 15 amino acids of the protein, might be suitable formeasuring the UL54/UL102 interaction, we tested whether antiserum 113,directed against the C-terminal 15 amino acids of HSV-1 UL30 (POL) couldbe used to detect the HSV-1 UL30/UL8 interaction. As a control we usedthe UL30-specific MAb 13185 that had previously been used to monitor theHSV-1 interaction. The assay was performed as described previously.Briefly, plates were coated with UL8 and bound UL30 was detected withMAb 13185 followed by HRP-conjugated anti mouse IgG and chromogenicsubstrate. The absorbance was recorded at 405 nm. The results are shownin FIG. 16 and are presented in groups of 4 bars. Bar 1 shows absorbancein wells with both UL8 and UL30, bars 2, 3 and 4 show the absorbancewhen UL30, UL8 or both UL8 and UL30 respectively were omitted. Data ingroups A and B show the results with and without MAb 13815, and confirmour previous findings showing that the signal is dependent on thepresence of HSV-1 UL8, UL30 and MAb 13185. Data in groups C, D, E and Fshow similar experiments in which the detecting antibody was rabbitserum 113 diluted 10³-, 10⁴- or 10⁵-fold or-omitted (No RAb). Boundantibody was detected with HRP-conjugated protein A. The data show thatin this assay, the signal is dependent on the presence of HSV-1 UL8,UL30 and the C-terminal antiserum 113. Thus, this C-terminal antiserumcan be used to monitor the HSV-1 UL30/UL8 interaction.

These findings suggested that the rabbit serum 144, specific for theC-terminus of HCMV UL54 might enable the HCMV UL54/UL102 interaction tobe monitored. The data presented in FIG. 14 indicated that this serumcould be diluted 5-fold and give an acceptable signal with UL54 bounddirectly to wells, and the antibody was accordingly used at thatconcentration. For the interaction assay, wells were coated with amountsof UL102 ranging from zero to 0.4 μg per well. UL54 was added in amountsranging from ranging from zero to 0.6 μg per well. The results (FIG. 17)show that the signal: 1) is dependent on the presence of both proteins,2) increases as the amount of UL102 is increased and 3) increases as theamount of UL54 is increased, up to 0.4 μg per well. We interpret thesedata as evidence for an interaction between UL54 and UL102.

Inhibition of the UL54/UL102 interaction by UL102 peptides. We wished todemonstrate that peptides at or near the C-terminus of other herpesvirushomologues of HSV-1 UL8 would disrupt the interaction between thehomologues of UL8 and POL. To do this, the peptides listed in Table 4were tested for their ability to block the interaction of HCMV UL102with HCMV UL54. Peptides, diluted in 100 mM Tris-HCl (pH 8.0) plus 0.1%Tween 20, together with 0.4 μg UL54, were added to microtiter wellspre-coated overnight with 0.4 μg UL102. The amount of UL54 bound wasdetermined after 1 h by measuring the absorbance at 405 nm as described.The absorbance in the absence of any peptide, 1.061±0.033, wasdetermined from six wells. The background absorbance in the absence ofany UL54, 0.240±0.025, was also determined from six wells and wassubtracted from all values. FIG. 18 shows the results, derived from theaverage of duplicate wells, for peptides 1 and 2 and a control peptide,RT85. The concentration of each peptide required to reduce UL54-bindingby 50% (the IC₅₀ value) was determined and is listed in Table 4.

TABLE 1 Properties of UL8-specific monoclonal antibodies Immunologicalreactivity^(a) MAb Immuno- Immuno- Inhibition of Desig- precipi- fluor-Western Blot POL/ULS nation ELISA tation escence (and region)^(b)interaction 801 ++ ++ ++ − − 802 ++ − − − − 803 ++ − − − − 804 ++ ++ + +− 805 ++ ++ ++ − − 807 ++ − − − + 809 ++ − − ++ (region 2) − 811 ++ ++++ ++ (region 1) − 812 ++ − + + (region 1) − 813 ++ + ++ − − 814 ++ − +++ (region 2) + 815 ++ − + − − 817 ++ ++ ++ ++ (region 3) + 818 ++ ++ ++++ (region 3) + 819 ++ ++ ++ ++ (region 3) + 820 ++ ++ − − −^(a)Reactivity in the immunological assays is subjectively described as:++ strong; + detectable; − not detectable. ^(b)The epitopes recognisedby these antibodies were mapped to the following locations within UL8:region 1, amino acids 165-253; region 2, amino acids 470-671; region 3,amino acids 717-750.

TABLE 2 Peptides used in this study Corresponding IC₅₀ ^(b) Peptideresidues in UL8 Sequence M_(r)a (μM) 1 739-750                         YPFDDKMSFLFA 1480 >250 2 728-750              AGVWGEGGKFVYPFDDKMSFLFA 2567 >250 3 726-750            VLAGVWGEGGKFVYPFDDKMSFLFA 2779 >250 4 724-750          TGVLAGVWGEGGKFVYPFDDKMSFLFA 2937 >250 5 722-750        VFTGVLAGVWGEGGKFVYPFDDKMSFLFA 3184 66 ± 22 6 724-735          TGVLAGVWGEGGKFV 1475 >250 7 719-738      IELVFTGVLAGVWGEGGKFV2077 2.3 ± 2.2 8 714-728 EILREIELVFTGVLA 1701 >250 7J^(c) —IVEFLKVGFGTEGGVWLVAG 2077  >20 RT85^(d) — VKLWYQLEKEPIVGA 1772 >250^(a)Relative molecular mass ^(b)The concentration of peptide required toreduce POL binding by 50% ^(c)The same amino acids as peptide 7 but injumbled order ^(d)Residues 423-437 of the reverse transcriptase of HIV-1(strain LAI)

TABLE 3 Properties and epitope mapping of POL-specific monoclonalantibodies Immunological reactivity Western Blot (and UL30 Deduced MAbImmuno- Immuno fragment Pattern of epitope designation ELISAprecipitation fluorescence recognised)^(a) reactivity^(b) location^(c)13088 + − − +(3) D 317-596 13112 + − − − E 13129 + + − + (2) B 213-30713185 + + + + (5) A  976-1071 13429 + + + + (5) A  976-1071 13455 + − +− F 13460 + − − + (2) D 213-307 13479 + + − − C 13488 + + − + (2) B213-307 13509 + + − − C 13528 + + − + (3) B 317-596 13579 + − − − E13584 + − − − E 13628 + + + + (6) A 1120-1127 ^(a)The numbers inparentheses denote the UL30 fragment recognised. The amino acids presentin the fragments are: fragment 1, 1-212; fragment 2, 162-316; fragment3, 308-658; fragment 4, 597-975; fragment 5, 875-1119; fragment 6,1072-1145 and fragment 7, 1128-1235 respectively. ^(b)Based on thepresence or absence of reactivity in immunoprecipitation,immunofluorescence and western blotting assays (see discussion).^(c)Based on western blot reactivity with fragments of the UL30expressed in E. coli.

TABLE 4 Peptides used in Example 3 Corresponding residues in IC₅₀ ^(b)Peptide UL102 Sequence M_(r) ^(a) (μM) 1 844-873^(d)      DEWRSLAVDAQHAAKRVASECLRFFRLNA 3413 40 2 838-863 TWLEERDEWRSLAVDAQHAARRVAS 3052 45 RT85^(c) VKLWYQLEKEPIVGA 1772 >200^(a)Relative molecular mass ^(b)The concentration of peptide required toreduce HCMV UL54-binding by 50% ^(c)Residues 423-437 of the reversetranscriptase of HIV-1 (strain LA1) ^(d)Contains R to K substitution atresidue 859

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18 1 12 PRT Artificial Sequence Description of ArtificialSequencepeptide derived from herpes simplex virus 1 Tyr Pro Phe Asp AspLys Met Ser Phe Leu Phe Ala 1 5 10 2 23 PRT Artificial SequenceDescription of Artificial Sequencepeptide derived from herpes simplexvirus 2 Ala Gly Val Trp Gly Glu Gly Gly Lys Phe Val Tyr Pro Phe Asp Asp1 5 10 15 Lys Met Ser Phe Leu Phe Ala 20 3 25 PRT Artificial SequenceDescription of Artificial Sequencepeptide derived from herpes simplexvirus 3 Val Leu Ala Gly Val Trp Gly Glu Gly Gly Lys Phe Val Tyr Pro Phe1 5 10 15 Asp Asp Lys Met Ser Phe Leu Phe Ala 20 25 4 27 PRT ArtificialSequence Description of Artificial Sequencepeptide derived from herpessimplex virus 4 Thr Gly Val Leu Ala Gly Val Trp Gly Glu Gly Gly Lys PheVal Tyr 1 5 10 15 Pro Phe Asp Asp Lys Met Ser Phe Leu Phe Ala 20 25 5 29PRT Artificial Sequence Description of Artificial Sequencepeptidederived from herpes simplex virus 5 Val Phe Thr Gly Val Leu Ala Gly ValTrp Gly Glu Gly Gly Lys Phe 1 5 10 15 Val Tyr Pro Phe Asp Asp Lys MetSer Phe Leu Phe Ala 20 25 6 15 PRT Artificial Sequence Description ofArtificial Sequencepeptide derived from herpes simplex virus 6 Thr GlyVal Leu Ala Gly Val Trp Gly Glu Gly Gly Lys Phe Val 1 5 10 15 7 20 PRTArtificial Sequence Description of Artificial Sequencepeptide derivedfrom herpes simplex virus 7 Ile Glu Leu Val Phe Thr Gly Val Leu Ala GlyVal Trp Gly Glu Gly 1 5 10 15 Gly Lys Phe Val 20 8 15 PRT ArtificialSequence Description of Artificial Sequencepeptide derived from herpessimplex virus 8 Glu Ile Leu Arg Glu Ile Glu Leu Val Phe Thr Gly Val LeuAla 1 5 10 15 9 20 PRT Artificial Sequence Description of ArtificialSequencepeptide derived from herpes simplex virus 9 Ile Val Glu Phe LeuLys Val Gly Phe Gly Thr Glu Gly Gly Val Trp 1 5 10 15 Leu Val Ala Gly 2010 15 PRT Artificial Sequence Description of Artificial Sequencepeptidederived from herpes simplex virus 10 Val Lys Leu Trp Tyr Gln Leu Glu LysGlu Pro Ile Val Gly Ala 1 5 10 15 11 30 PRT Artificial SequenceDescription of Artificial Sequencepeptide derived from herpes simplexvirus 11 Asp Glu Trp Val Arg Ser Leu Ala Val Asp Ala Gln His Ala Ala Lys1 5 10 15 Arg Val Ala Ser Glu Gly Leu Arg Phe Phe Arg Leu Asn Ala 20 2530 12 26 PRT Artificial Sequence Description of ArtificialSequencepeptide derived from herpes simplex virus 12 Thr Trp Leu Glu GluArg Asp Glu Trp Val Arg Ser Leu Ala Val Asp 1 5 10 15 Ala Gln His AlaAla Arg Arg Val Ala Ser 20 25 13 15 PRT Artificial Sequence Descriptionof Artificial Sequencepeptide derived from herpes simplex virus 13 HisLeu Glu Pro Ala Phe Leu Pro Tyr Ser Val Lys Ala His Glu 1 5 10 15 14 15PRT Artificial Sequence Description of Artificial Sequencepeptidederived from herpes simplex virus 14 Val Leu Ser Ser Ala Leu Pro Ser ValThr Ser Ser Ser Ser Gly 1 5 10 15 15 30 DNA Artificial SequenceDescription of Artificial Sequence primer 15 attatctaga ccgctatgtttttcaacccg 30 16 31 DNA Artificial Sequence Description of ArtificialSequence Primer 16 tatatctaga catcatcacc gtccccagtc a 31 17 31 DNAArtificial Sequence Description of Artificial Sequence Primer 17attaggatcc ttctgtccga ggatgaccgc t 31 18 28 DNA Artificial SequenceDescription of Artificial Sequence Primer 18 attaggatcc acgtcacacgctaagagc 28

What is claimed is:
 1. An antiviral agent which prevents or hindersreplication of a herpesvirus in vitro by specifically binding to POL orUL8, thus inhibiting the association between UL8 and POL, wherein “UL8”is defined as UL8 of HSV-1 or the homologues thereof in otherherpesviruses and “POL” is defined as POL of HSV-1 or homologues thereofin other herpesviruses, wherein said agent is a peptide selected fromthe group of peptides: a) VFTGVLAGVWGEGGKFVYPFDDKMSFLFA (SEQ ID NO: 5);b) IELVFTGVLAGVWGEGGKFV (SEQ ID NO: 7); c)DEWVRSLAVDAQHASKRVASEGLRFFRLNA (SEQ ID NO: 11) and;TWLEERDEWVRSLAVDAQHAARRVAS (SEQ ID NO: 12).
 2. An antiviral agent asclaimed in claim 1 which is a synthetic peptide.
 3. A method ofpreventing replication of a herpesvirus, said method comprisingproviding an agent able to bind specifically to UL8 or POL therebyinhibiting the association between UL8 and POL in vitro, wherein “UL8”is defined as UL8 of HSV-1 or the homologues thereof in otherherpesviruses and “POL” is defined as POL of HSV-1 or the homologuesthereof in other herpesvirus, and wherein said agent is a peptideselected from the group of peptides: a) VFTGVLAGVWGEGGKFVYPFDDKMSFLFA(SEQ ID NO: 5); b) IELVFTGVLAGVWGEGGKFV (SEQ ID NO: 7); c)DEWVRSLAVDAQHASKRVASEGLRFFRLNA (SEQ ID NO: 1) and;TWLEERDEWVRSLAVDAQHAARRVAS (SEQ ID NO: 12); said method comprisingadding said agent to said replicating herpesvirus in sufficient quantityto cause said inhibition and monitoring the effect on viral replicationand thus determining the presence or extent of said inhibition.